Mechanism Of H₂S Mediated Cadmium Regulation ROS Inhibites Non-heading Chinese Cabbage Root Growth

The plants under heavy metal cadmium stresses will be causing huge outbreak of ROS,which produces oxidative damage to plant growth.In Arabidopsis thaliana,the study found that the balance of two kinds of reactive oxygen species(hydrogen peroxide,H2O2 and superoxide radical O2·-)in the root tip plays an important role in the growth of plant root tip,and the balance is broken causing inhibiting the growth of plant root.Also,studies have proved that adding exogenous H2S can alleviate plant physiological toxic under Cd environment.On the basis of two kinds of theories in the study,whether physiological effect of the growth inhibition by Cd pollution is caused by breaking the active oxygen balance in the root tip,and whether endogenous H2S under cadmium pollution was also involved in regulating the active oxygen balance and its role in path.The main results are as follows:Firstly,different cadmium concentrations(4 ?pM and 16 ?M)treatment with Brassica rapa root inhibits the growth of root length,root inhibition rate are 23%and 53%,respectively.But 4 ?M Cd treatment didn't cause oxidative damage and cell death,while 16?M causes this response;4 ?M Cd brokes the balance between of H2O2 and O2·-in root tip,causing rising the root tip H2O2 content,and decreasing O2·-content,the effect also having time gradient effect.Adding the exogenous NADPH oxidase inhibitors DPI removing O2·-and exogenous H2O2 are processed with 4 ?M Cd the same phenotype.The results show that Brassica rapa root growth inhibition with 4 ?M cadmium treatment was caused by breaking reactive oxygen balance,and causing no oxidative damage in vitro.Secondly,in different time gradient experiment,4 ?M Cd treatment leads to the abundant accumulation of endogenous H2S in the root tip,and induced the Br LCDs and Br_DCDs gene expression up-regulation.In 4 ?Cd processing on the basis of adding the H2S synthetase inhibitors(DL-propargylglicine,PAG)and exogenous scavenger(Hypotaurine,HT),reduced the endogenous H2S content and H2O2 content in the root tip,and increased O2·-content,the results with simple 4 ?M Cd treatment showed a opposite trend.At the same time,the H2S content in root is falling and root length also recover appropriate length.The results show that the endogenous H2S in root was involved in regulating reactive oxygen species equilibrium state under cadmium stress to regulate the growth of root.Thirdly,reference to Arabidopsis root length experiment,UPB1 as a transcription factor can inhibit further to control several PODS.gene expression in the root tip balance between O2'-and H2O2.In Brassica rapa,we blast two UPB1 genes(Bra004465,Bra021395)and four POD genes(Bra035235,Bra033551,Bra006423,Bra023639).4?M Cd treatment induced Br_UPB1A and Br_UPBIB genes up-regulation,and stimulated the UPB1,POD genes down-regulation,on the basis of the 4 ?M Cd treatment adding PAG or HT can be reversed by 4 ?M Cd treatment,showing that H2S by means of participating in regulating UPBl and the expression of target genes,consequently affecting the balance of ROS to regulate the root long growth.Finally,cloning and bioinformatics method analysis the Br_UPB1A gene in Brassica rapa,Br_UPB1A gene length is 309 bp,it encods 102 amino acids without introns distribution and has a conservative bHLH structure domain made up of 56 amino acids typical helix transcription factors.And due to the lack of Glutamic acid(Glu,E-13)and Arginine(Arg,R-16),it belongs to the atypical bHLH transcription factors,namely non-DNA-binding protein.This kind of transcription factors is mainly with the rest of the bHLH transcription factor forming dimers to exercise the function,however through database retrieval failed to find about Br_UPB1A and its homologous protein AtUPB1 protein interactions.The Br_UPB1A protein belongs to hydrophilic protein,its secondary structure is mainly composed of alpha helix(helix)and random curl(coil),scattered on the whole Br_UPB1A protein structure.